Stabilization of a Multimeric β-Galactosidase from Thermus sp. Strain T2 by Immobilization on Novel Heterofunctional Epoxy Supports Plus Aldehyde-Dextran Cross-Linking

• Published in: Biotechnology progress 2004, Vol.20 (1), p.388-392
• Main Authors: Pessela, Benevides C. C., Mateo, Cesar, Fuentes, Manuel, Vian, Alejandro, García, José L., Carrascosa, Alfonso V., Guisán, José M., Fernández-Lafuente, Roberto
• Format: Article
• Language: English
• Subjects: Adsorption; Aldehydes - chemistry; beta-Galactosidase - chemistry; Biological and medical sciences; Biotechnology; Dextrans - chemistry; Dimerization; Enzyme Activation; Enzyme Stability; Enzymes, Immobilized - chemistry; Epoxy Compounds - chemistry; Fundamental and applied biological sciences. Psychology; Hydrolysis; Lactose - chemistry; Polymers - chemistry; Protein Conformation; Protein Structure, Quaternary; Species Specificity; Thermus; Thermus - classification; Thermus - enzymology
• ISSN: 8756-7938; 1520-6033
• DOI: 10.1021/bp034183f

This work exemplifies the advantages of using a battery of new heterofunctional epoxy supports to immobilize enzymes. We have compared the performance of a standard Sepabeads‐epoxy support with other Sepabeads‐epoxy supports partially modified with boronate, iminodiacetic, metal chelates, and ethylenediamine in the immobilization of the thermostable β‐galactosidase from Thermus sp. strain T2 as a model system. Immobilization yields depended on the support, ranging from 95% using Sepabeads‐epoxy‐chelate, Sepabeads‐epoxy‐amino, or Sepabeads‐epoxy‐boronic to 5% using Sepabeads‐epoxy‐IDA. Moreover, immobilization rates were also very different when using different supports. Remarkably, the immobilized β‐galactosidase derivatives showed very improved but different stabilities after favoring multipoint covalent attachment by long‐term alkaline incubation, the enzyme immobilized on Sepabeads‐epoxy‐boronic being the most stable. This derivative had some subunits of the enzyme not covalently attached to the support (detected by SDS‐PAGE). This is a problem if the biocatalysts were to be used in food technology. The optimization of the cross‐linking with aldehyde‐dextran permitted the full stabilization of the quaternary structure of the enzyme. The optimal derivative was very active in lactose hydrolysis even at 70 °C (over 1000 IU/g), maintaining its activity after long incubation times under these conditions and with no risk of product contamination with enzyme subunits.